Potentiating effects of clofibrate on prostaglandin-dependent and -independent pathways of human platelet activation: evidence for involvement of cyclic AMP

Although clofibrate has been shown to inhibit platelet aggregation that is caused by thrombin, ADP and epinephrine, by blocking the release of arachidonic acid from platelet phospholipids [8], here we have demonstrated that clofibrate enhanced platelet aggregation by arachidonic acid and PLC and reversed the effects of PGE1 on platelet cAMP concentration and on PLC-induced secretion of [14C]-5HT in similar, concentration-dependent manners. Taken together, these findings strongly suggest that the proaggregatory effect of clofibrate is mediated by a lowering of cAMP in platelets.     

Effects of clofibrate and 6-substituted chroman analogs on human platelet function:: Mechanism of inhibitory action

The effects of cloflbrate (CPIB) and two related cyclic analogs, 6-chlorochroman-2-carboxylic acid (CCCA) and 6-phenylchroman-2-carboxylic acid (PCCA), on human platelet function were evaluated. CPIB, CCCA and PCCA all inhibited platelet activation, i.e. aggregation and secretion of [¹⁴C]serotonin induced by ADP, epinephrine, collagen and thrombin, in a concentration-dependent manner. PCCA was at least fifty-two times more effective as an inhibitor of ADP-, epinephrine- and collagen-induced platelet activation and only 2-fold more effective as an inhibitor of thrombin-induced platelet activation when compared with CPIB or CCCA. Only PCCA inhibited platelet aggregation and [¹⁴C]serotonin secretion induced by arachidonic acid (AA) in a concentration-dependent manner. CPIB and CCCA did not inhibit AA-induced platelet activation. In fact, both of these agents had a potentiating effect on the onset of platelet aggregation by AA. All three compounds inhibited thrombin-induced release of [³H]arachidonic acid ([³H]AA) from platelet phospholipids and thrombin-mediated malondialdehyde (MDA) production. Only PCCA, however, inhibited AA-induced MDA production. These results indicate that CPIB, CCCA and PCCA all inhibit platelet activation by inhibiting prostaglandin biosynthesis. PCCA blocked AA-induced platelet activation, and this additional inhibitory action of PCCA appears to be responsible for its comparatively higher inhibitory potency. A comparison of the structure-activity relationship of the inhibitors indicated that replacement of the chloro group by a phenyl group produced a compound (PCCA) that was a potent inhibitor of prostaglandin biosynthesis and was thereby a more effective antiaggregatory agent than either CPIB or CCCA.     

Reduction in cutaneous and hepatic glutathione contents,glutathione S-transferase and aryl hydrocarbon hydroxylase activities following topical application of acrylamide to mouse

A single topical application of acrylamide to mouse resulted in time-dependent depletion in cutaneous and hepatic glutathione contents, glutathione S-transferase and aryl hydrocarbon hydroxylase activities.     

Effects of ethanol and barbiturates on Ca2+-ATPase activity of erythrocyte and brain membranes

Exposure to ethanol or pentobarbital in vitro stimulated the ATP-dependent efflux of calcium from human red blood cells (RBC) and the Ca2+-ATPase activity of RBC and rat brain synaptic plasma membranes (SPM). These effects were obtained with concentrations of ethanol (50 mM) and pentobarbital (60 microM) associated with intoxication in vivo. The enhancement of SPM Ca2+-ATPase by ethanol was due to an increase in the apparent affinity of the enzyme for calcium with no change in the maximum velocity. SPM Ca2+-ATPase was also stimulated by an unsaturated fatty acid, cis-vaccenic acid methyl ester (cis-VAME). The membrane-disordering effects of ethanol, four barbiturates and cis-VAME were evaluated in SPM using the fluorescent probe molecule 1,6-diphenyl-1,3,5-hexatriene (DPH). All the compounds decreased the fluorescence polarization of DPH, and these decreases were proportional to the increase in Ca2+-ATPase produced by these drugs. These findings suggest that the increase in Ca2+-ATPase and calcium efflux produced by ethanol and pentobarbital results from the membrane-disordering effects of these drugs.     

Effects of hypophysectomy on acetylcholinesterase and butyrylcholinesterase in the rat

Acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) activities were examined in several tissues of normal and hypophysectomized male and female rats. Significant sex differences in the mean AChE activities of normal rats were observed in the superior cervical ganglion (three times more activity in males) and in serum (50% more activity in females). Sex differences in the BuChE activity of serum and liver were even larger (ten times more activity in females), but the activity of other tissues was similar in both sexes. Hypophysectomy had little effect on the mean activity of AChE but did alter BuChE activity in certain tissues. Most of the effects of hypophysectomy on mean BuChE activity were opposite in direction in the two sexes. For example, in males hypophysectomy caused increases in the BuChE activity of serum (300%) and liver (43%), while in females it caused decreases in both tissues (25 and 30% respectively). In rats of a given group, the AChE activity of each tissue appeared to be regulated independently of the activity in other tissues. By contrast, BuChE activity showed statistically significant correlations in more than half of the tissue-pairs examined in control rats of either sex. These correlations can be considered to reflect a tendency toward body-wide regulation. In female rats, the cross-tissue correlations were largely eliminated by hypophysectomy. This finding indicates that the regulation of BuChE mav be strongly affected by hormones under the control of the pituitary gland. However, in male rats, only the correlations involving atria were altered by hypophysectomy. Therefore, the effects of hormones on BuChE are probably both sex and tissue dependent.     

Effects of ethanol on fatty acid composition of muscle phospholipids of rats fed nutritionally complete liquid diets

Although the effects of ethanol on phospholipid fatty acid composition have been examined in single cell organisms and in rodents given ethanol as a vapor or in solution as sole drinking fluid, there are no reports of analogous experiments with nutritionally adequate liquid diets as the source of ethanol. In this experiment, young, 88 ± 5 g, male Sprague-Dawley rats given a nutritionally adequate liquid diet containing ethanol voluntarily consumed 12–18 g ethanol per kg body weight per day after 23 days. In 27 days with food intakes 64% of control, weight gains of ethanol-fed animals (group 2) were 50% of ad lib.-fed control animals (group 1) but 88% of isoenergetically pair-fed animals (group 3). Thus, energy in the ethanol diet was utilized for growth 88% as efficiently as isoenergetic diets containing dextrin. Liver and gastrocnemius muscle weights of group 2 were significantly lower than group 3 but brain weights were not similarly affected. Blood ethanol levels determined on days 14 and 24 were above 200 mg/dl at 10:00 p.m. and 7:00 a.m. but decreased to 50 mg/dl at 4:00 p.m. on a feeding schedule that began at 5:00 p.m. Dependence was confirmed by withdrawal symptomology. Compared to group 1, phospholipids isolated from gastrocnemius muscles of group 2 exhibited significant modifications in fatty acid composition. In ethanol-fed animals, 18:0 and 20:4 were lower, and 18:1 and 18:2 were higher, than ad lib.-fed controls. However, when group 2 was compared to group 3, the pair-fed control, there was no significant difference in fatty acid composition. The observed changes in fatty acid composition appear to have been due to the reduced food consumption that accompanied the model rather than to ethanol per se. These findings underline the importance of appropriate controls in liquid diet animal models of alcoholism.     

The interaction between benzodiazepine antagonists and barbiturate-induced cerebrovascular and cerebral metabolic depression

It has been reported that pentobarbital facilities binding to benzodiazepine receptors binding at anesthetic concentrations and that this action may play a role in the anesthetic potency of this barbiturate. The interaction between pentobarbital and benzodiazepine receptors was tested with Ro 15-1788 which is reported to be a pure benzodiazepine antagonist and 3-hydroxymethyl-beta-carboline (3-HMC), an antagonist which has inverse activity alone. Cerebral blood flow (CBF) and cerebral oxygen consumption (CMRO2) were measured in rats after injections of pentobarbital with and without the antagonists. Pentobarbital produced dose-dependent decreases in cerebral blood flow and cerebral oxygen consumption at 15 and 30 mg/kg. The antagonist Ro 15-1788 (10 mg/kg) stimulated cerebral blood flow and cerebral oxygen consumption alone but did not alter the cerebral depression produced by pentobarbital. The cerebral metabolic stimulation produced by Ro 15-1788 was unexpected since the drug is reported to be a pure antagonist without agonistic activity, but the lack of effect on pentobarbital-induced cerebral depression is consistent with other reports. 3-Hydroxymethyl-beta-carboline at 10 mg/kg did not stimulate cerebral blood flow and cerebral oxygen consumption but significantly antagonized the decrease in cerebral oxygen consumption produced by 15 mg/kg pentobarbital. 3-Hydroxymethyl-beta-carboline had no significant effect on decreases in cerebral blood flow and cerebral oxygen consumption produced by phenobarbital, a barbiturate which is reported not to alter binding to benzodiazepine receptors. The ability of 3-HMC to antagonize the effects of pentobarbital would be consistent with an action of both drugs at the benzodiazepine receptor but not by altering binding to an endogenous receptor.     

A correlation between hydroxyl radical generation and ethanol oxidation by liver, lung and kidney microsomes

A comparison of the abilities of microsomes from liver, kidney and lung to oxidize ethanol and to generate hydroxyl radicals was conducted to determine if these two variables correlated with one another. The oxidation of 2-keto-4-thiomethylbutyric acid (KTBA) to ethylene, and the production of formaldehyde from dimethylsulfoxide (Mc₂SO), served as chemical probes for the detection of the production of hydroxyl radicals by the microsomes. Liver microsomes oxidized ethanol at rates several-fold greater than those found with lung and kidney microsomes. This greater rate of ethanol oxidation by liver microsomes correlated with a greater rate of oxidation of the hydroxyl radical scavengers by the liver microsomes (liver > lung ≈ kidney). In all tissues, the addition of azide, an inhibitor of catalase, augmented the rate of oxidation of Me₂SO and KTBA. The addition of iron-EDTA (a OH-potentiating agent) increased the rates of oxidation of ethanol by the microsomes from the three tissues. This increase again correlated with an increase in the oxidation of Me₂SO and KTBA. The greater rate of oxidation of ethanol and the hydroxyl radical scavengers by liver microsomes may reflect the relative specific content of cytochrome P-450 (6- to 12-fold greater) and specific activity of NADPH-cytochrome c reductase (4-fold greater) in liver as compared to lung and kidney microsomes. Relative turnover numbers (units per nmole cytochrome P-450) demonstrated equivalent activities for liver and kidney, whereas lung had a higher turnover number for ethanol oxidation and hydroxyl radical generation. These data support the hypothesis that the oxidation of ethanol by microsomes may be mediated by the relative capacity of the microsomes to generate hydroxyl radicals during microsomal electron transport, which in turn may be related to the relative content and/or activities of the components of the electron transport chain.     

Effects of rubratoxin B on the hepatic monooxygenase system in phenobarbital and 3-methylcholanthrene induced male mice

Alterations in the hepatic microsomal monooxygenase system and in the concentrations of rubratoxin B in urine and feces were examined in male mice pretreated with corn oil, phenobarbital or 3-methylcholanthrene (3MC) and then given a single i.p. dose of rubratoxin B (1 mg/2.5 ml propylene glycol/kg). Twenty-four hours later the following parameters were determined: hepatic cytochrome P-450 content, enzyme activities of NADPH-cytochrome c reductase, NADPH-dependent dehydrogenase, aniline hydroxylase and ethylmorphine N-demethylase, and hapatic microsomal protein and reduced glutathione levels. Excretion of rubratoxin B in urine and feces also was determined. Rubratoxin B reduced the elevated cytochrome P-450 (136%, 134%) and protein (128%, 112%) to control values in animals pretreated with phenobarbital or 3MC, respectively; whereas, in the corn oil pretreated group, the mycotoxin reduced cytochrome P-450 by 38%. Aniline hydroxylase activity was reduced 31% or more in all pretreated animals. Rubratoxin B did not affect ethylmorphine N-demethylase activity in mice pretreated with phenobarbital; however, the enzyme activity was decreased significantly in the 3MC group. Rubratoxin B reduced the hepatic glutathione level in animals receiving 3MC (33%) or corn oil (22%). More rubratoxin B was detected in the urine than in the feces regardless of pretreatment. Only trace amounts of toxin were detected in the feces of animals from the 3MC group. These data suggest a greater effect of rubratoxin B in the 3MC pretreated mice than in the phenobarbital animals.     

Influence of harmaline on the ability of pargyline to alter catecholamine metabolism in rats

When pargyline hydrochloride (20 mg/kg, i.p.) was injected into rats 48 hr before the measurement of monoamine oxidase (MAO) activity, the oxidation of [¹⁴C]phenylethylamine (type B MAO) and of [¹⁴C]-serotonin (type A MAO) was inhibited. Neither type A nor type B MAO was inhibited 48 hr after the injection of harmaline hydrochloride (30mg/kg, i.p.) a short-acting, reversible, highly selective inhibitor of type A MAO. When harmaline was given just before pargyline, it prevented the inhibition of type A MAO by pargyline but not the inhibition of type B MAO. Pargyline alone elevated epinephrine, norepinephrine, and dopamine concentrations in brain regions and norepinephrine concentration in heart. The concentration of dopamine metabolites (3,4-dihydroxyphenylacetic acid and homovanillic acid) was decreased. Pretreatment with harmaline prevented all of these effects of pargyline. The findings suggest that inhibition of type A MAO is involved in the inhibition of catecholamine metabolism by pargyline, since harmaline pretreatment did not prevent inhibition of type B MAO and would not be expected to alter any other possible actions of pargyline. These findings support the idea that type A MAO is primarily responsible for the oxidation of epinephrine, norepinephrine, and dopamine in rat brain and of norepinephrine in rat heart.     

Effects of pentobarbital and Ca2+ on the resting and K+-stimulated release of several endogenous neurotransmitters from rat midbrain slices

The release of endogenous amino acids [γ-aminobutyric acid (GABA), glutamate, aspartate, glycine and alanine] and of 5-hydroxytryptamine (5-HT) and acetylcholine (ACh) from rat midbrain slices was examined under various conditions of superfusion. Depolarization with high K⁺ stimulated the release of all substances examined, but the K⁺-stimulated release was Ca²⁺ dependent only for GABA, glutamate, aspartate, 5-HT and ACh. Pentobarbital, although not substantially affecting resting release, inhibited the K⁺stimulated release of GABA, glutamate, aspartate and ACh markedly and significantly. The effect of pentobarbital on K⁺-stimulated 5-HT release was not statistically significant in this series of experiments. These results are consistent with the hypothesis that the barbiturate inhibits stimulated transmitter release by inhibiting Ca²⁺ influx during depolarization. The tissue content of amino acids and 5-HT decreased considerably from fresh tissue levels after 50 min of perfusion in regular low K⁺ medium; in contrast, ACh tissue levels increased slightly during this time. K⁺ stimulation resulted in an increased synthesis particularly of GABA, glutamate, glycine, alanine and ACh, for the amount released into the medium was far more than that lost from the tissue. 5-HT and possibly aspartate, on the other hand, were released into the medium on stimulation largely at the expense of tissue stores, under our experimental conditions.     

Further studies on the increase in drug-metabolizing capacity adjacent to intrahepatic Morris hepatomas

Microsomal cytochrome P-450 content was higher in histologically non-tumorous liver adjacent to intrahepatically implanted Morris hepatomas 5123D or 7795 than in histologically normal liver far removed from each tumor. V_max values for microsomal benzo[a]pyrene monooxygenase activity and cyclophosphamide activation were also significantly higher in tumor-adjacent liver than in normal liver far removed from tumor. K_m values of these reactions were unchanged. After intrahepatic implantation, inert spheres of several different materials produced no regional differences in hepatic microsomal cytochrome P-450 content. Both intrahepatic Morris hepatomas exhibited markedly reduced cytochrome P-450 content and benzo[a]pyrene monooxygenase activity. Cyclophosphamide biotransformation could not be detected in microsomes from either Morris hepatoma. Similar recoveries from microsomes of far-removed and tumor-adjacent liver indicated that differences between these regions in drug-metabolizing activity could not be attributed to different stabilities or sedimenting properties of their microsomes. Although microsomal recovery was significantly less from hepatomas than from far-removed or tumor-adjacent liver, this loss of tumor microsomes accounted for only a small part of the reductions in cytochrome P-450-mediated monooxygenases observed within tumors. Compared to control rats. tumor-bearing rats exhibited no change in hepatic drug-metabolizing capacity measured in vivo by hexobarbital sleeping times and antipyrine elimination rates. Phenobarbital (PB) pretreatment of tumor-bearing rats induced cytochrome P-450 to different extents within far-removed liver, tumoradjacent liver, and both hepatomas. The same differential inducibility occurred with PB pretreatment for cyclophosphamide activation. After PB induction, differences in drug-metabolizing activity between far-removed and tumor-adjacent liver disappeared; though induced, these activities remained lower in the hepatomas than in other regions. These changes in drug-metabolizing activity in both basal and PB-induced states of various hepatic regions were related to changes in cellularity of tumor-adjacent tissue. Hepatocellular nuclei prepared from tumor-containing liver were separated into diploid and tetraploid classes by sucrose density gradient centrifugation. Compared to far-removed liver, tumoradjacent liver contained significantly more diploid nuclei and less tetraploid nuclei.     

Effects of ethanol on arrhenius parameters and activity of mouse striatal adenylate cyclase

Arrhenius plots of basal and dopamine (DA)-stimulated adenylate cyclase activities exhibited discontinuities at 20°, while the plot of fluoride-stimulated adenylate cyclase activity was linear over the studied temperature range. None of the Arrhenius parameters were altered by in vitro addition of ethanol (75 or 750 mM) to enzyme assay mixtures, and Arrhenius parameters were found to be unchanged when enzyme obtained from animals rendered tolerant to, and physically dependent on, ethanol was assayed. The differences between the response to ethanol of adenylate cyclase and the response of other membrane-bound enzymes [e.g. (Na⁺-K⁺ATPase], as measured by Arrhenius plots, may indicate different sites of action of ethanol. When the specific activity of adenylate cyclase was examined, ethanol was found to stimulate activity at all temperatures tested. The dose-response curve for ethanol activation of basal adenylate cyclase activity was shifted to the right for enzyme obtained from mice chronically treated with ethanol. Analysis of the data indicated that activation of adenylate cyclase by ethanol (as well as by DA) was an entropy-driven process. Since ethanol treatment did not affect the Arrhenius parameters, which appear to be associated with membrane lipids, it is suggested that enzyme activation by ethanol results from direct effects on the enzyme or regulatory protein. Resistance to this effect occurs through changes in protein conformation following chronic ethanol treatment.     

Characterization and solubilization of the specific binding sites for d-α-tocopherol from human erythrocyte membranes

Previous work from our laboratory has demonstrated the presence of specific binding sites for d-α-tocopherol (vitamin E) in intact human erythrocytes [A. E. Kitabchi and J. Wimalasena, Biochim. biophys. Acta684, 300 (1982)]. The binding was time, temperature and cell concentration dependent. To localize the binding sites, red blood cells were further fractionated; greater than 90% of the tocopherol binding sites were localized on membranes. The washed membrane fraction from normal human erythrocytes has specific binding sites for d-α-tocopherol with properties suggestive of protein receptors. Two binding sites with K_a values of 3.31 × 10⁷ M^?1 and 1.51 × 10⁶ M^?1 were demonstrated, and solubilized d-α-tocopherol binding site complexes were resolved to major component with an M_r of 65,000 and a minor component with an M_r of 125,000.     

Epoxide hydrolase activity in the mitochondrial and submitochondrial fractions of mouse liver

Distribution of epoxide hydrolase activity in subcellular fractions of livers from male Swiss-Webster mice and Sprague-Dawley rats was monitored with trans-β-ethylstyrene oxide, trans-stilbene oxide and benzo[a]pyrene 4,5-oxide following differential centrifugation. With the former two substrates the highest activity was encountered in the cytosolic fraction; however, significant activity was found in the mitochondrial fraction. These fractions hydrated benzo[a]pyrene 4,5-oxide very slowly, and the major benzo[a]pyrene 4,5-oxide hydrolyzing activity was recovered in the microsomal fraction. Using Triton WR-1339-treated mice, it was shown that trans-β-ethylstyrene oxide hydrolyzing activity was predominantly localized in the mitochondria rather than in lysosomes and peroxisomes. Subsequent separation of the mitochondrial fraction into submitochondrial components by swelling, shrinking, and sonication, followed by sucrose density gradient centrifugation, showed that most of the epoxide hydrolyzing activity was present in the matrix and intermembrane space fraction. Significant activity was also present in the outer and inner membrane fractions. However, epoxide hydrolyzing activity in these fractions was reduced if either increased sonication times were used or the fractions were washed, indicating possible contamination of these fractions by the matrix and intermembrane space enzyme(s). The epoxide hydrolase activity in the mitochondrial and cytosolic fractions in mice appeared similar with regard to inhibition, molecular weight, and substrate selectivity.     

Effects of benzene vapour on the gamma-aminobutyric acid (GABA) system in rat brain.

Chronic benzene intoxication (0.35 mg/l) initially caused an increased in GABA concentrations with a simultaneous decrease in the glutamic and aspartic acid levels in cerebellum and pons Varolii of rats. On the 14th day of chronic exposure the level of all three amino acid was increased and associated with a depressive character of EEG patterns throughout the cerebellum, pons Varolii, and the sensori-motorial region of the cerebral hemispheres. On the 30th day, the GABA level had almost returned to its initial level but concentrations of glutamic and aspartic acid were decreased by more than 60% in cerebellum and by 20% in pons Varolii. During acute benzene poisoning (35.0 mg/l, for 15 min) the level of GABA was increased 5–6 times above the normal values. A 45% increase in the GABA level and a decreased amount of glutamic and aspartic acid were still observed 48 hr after the cessation of benzene inhalation. GAD activity in cerebellum and pons Varolii, during acute and chronic benzene intoxication, was higher than that of untreated animals: it was greater on the 14th day of chronic intoxication, 5- and 2-fold, respectively, and 4- and 3-fold at the 15th minute of acute intoxication. The GABA-T activity was also increased in these two structures (4–5-fold) by low and high concentrations of benzene vapour.     

Effect of chronic ethanol consumption on aldehyde dehydrogenase activity in the baboon

Baboon liver has detectable aldehyde dehydrogenase (A1DH) activity in the mitochondrial, microsomal and soluble fractions. Based on kinetic data, the mitochondrial and soluble fractions each appear to contain two forms of A1DH, one with a high, and another with a low, K_m for acetaldehyde. In the microsomes there was activity only with millimolar concentrations of acetaldehyde. In the baboon liver, about 75 per cent of total A1DH activity resides in the mitochondria and 20 per cent in the soluble faction. Chronic ethanol consumption decreased total and low K_m mitochondrial A1DH activity in baboon liver. In rats, ethanol consumption also resulted in decreased mitochondrial low K_m A1DH activity.